Roitt's Essential Immunology

Syk fulfills a critical role within the B-cell activation process; disruption
of the gene encoding Syk in the mouse has profound effects on downstream
events in B-cell signaling and results in defective B-cell development. In
this respect, Syk serves a similar role in B-cells to that served by ZAP-70 in

T-cells. Active Syk phosphorylates and recruits BLNK (B-cell linker; also
called SLP65, BASH and BCA) to the BCR complex. Upon
phosphorylation by Syk, BLNK provides binding sites for phospholipase

Cγ2 (PLCγ2), Btk and Vav. Recruitment of Btk in close proximity to PLCγ2
enables Btk to phosphorylate the latter and increase its activity. Just as in
the T-cell signaling pathway, activated PLCγ2 initiates a pathway that
involves hydrolysis of PIP to generate diacylg-lycerol and inositol
2
triphosphate and results in increases in intracellular calcium and PKC
activation (Figure 8.18). PKC activation, in turn, results in activation of the

NFκB and JNK transcription factors and increased intracellular calcium
results in NFAT activation, just as it does in T-cells.
The Vav family of guanine nucleotide exchange factors consists of at least
three isoforms (Vav-1,-2 and-3) and is known to play a crucial role in B-cell

signaling through activation of Racl and regulating cytoskeletal changes
after BCR cross-linking; Vav-1-deficient B-cells are defective in
proliferation associated with cross-linking of the BCR (Figure 8.18).

The BCR cross-linking model seems appropriate for an understanding of
stimulation by type 2 thymus-mdependent antigens, as their repeating
determinants ensure strong binding to, and cross-l inking of, multiple lg
receptors on the B-cell surface to form aggregates that persist owing to the

long half-l ife of the antigen and sustain the high intracellular calcium
needed for activation. On the other hand, type 1 T-independent antigens,
like the T-cell polyclonal activators, probably bypass the specific receptor

and act directly on downstream molecules such as diacylglycerol and
protein kinase C as lg-a and lg-ß are not phosphorylated.


Figure 8.18. Signaling cascade downstream of antigen-driven B-cell
receptor (BCR) ligation.
Upon interaction with antigen, the BCR is recruited to lipid rafts where
ITAMs within the lg-α/β heterodimer become phosphorylated by Lyn. This

is followed by recruitment and activation of the Syk and Btk kinases.
Phosphorylation of the B-cell adaptor protein, BLNK, creates binding sites
for several other proteins, including PLCγ2 that promotes PIP hydrolysis
2
and instigates a chain of signaling events culminating in activation of the
NFAT and NFκB transcription factors. The CD19 coreceptor molecule is
also phosphorylated by Lyn and can suppress the inhibitory effects of GSK3

on NFAT through the PI3K/Akt pathway. BCR stimulation also results in
rearrangement of the cell cytoskeleton via activation of Vav that acts as a
guanine nucleotide exchange factor for small G-proteins such as Rac and
Rho.























B-cells require co-stimulation via the B-cell co-

receptor complex for efficient activation


Similar to T-cells, B-cells also require two forms of co-stimulation to mount
efficient effector responses. One form of co-stimulation takes place at the
point of initial encounter of the BCR with its cognate antigen and is

provided by the B-cell coreceptor complex that is capable of engaging with
molecules such as complement that may be present in close proximity to the
specific antigen recognized by the BCR. The other form of co-timulation

required by B-cells takes place after the initial encounter with antigen and is
provided by T-cells in the form of membrane-associated CD40 ligand that

engages with CD40 on the B-cell. This form of co-stimulation requires that
the B-cell has internalized antigen, followed by processing and presentation
on MHC class II molecules to an appropriate T-cell. If the B-cell is
displaying an MHC–peptide combination recognized by the T-cell, the latter

will be stimulated to produce cytokines (such as IL-4) as well as provide
co-stimulation to the B-cell in the form of CD40L. We will consider the
nature of the co-stimulatory signals provided by the B-cell co-receptor
complex here and deal with CD40L-based co-stimulation in a separate

section below.

Figure 8.19. The B-cell coreceptor complex.
The B-cell coreceptor complex provides co-stimulatory signals for B-cell
activation through recruitment of a number of signaling molecules,

including phosphatidylinositol 3-kinase and Vav, which can amplify signals
initiated through the B-cell receptor. On mature B-cells, CD19 forms a
tetrameric complex with three other proteins: CD21 (complement receptor
type 2), CD81 (TAPA-1) and CD225 (interferon-induced transmembrane

protein 1) (LEU13). See also Figure 4.6.




























Thie mature B-cell coreceptor complex (Figure 8.19) is composed of four
components: CD19, CD21 (complement receptor type 2, CR2), CD81
(TAPA-1) and CD225 (LEU13, interferon-induced transmembrane protein

1). CR2 is a receptor for the C3d breakdown product of complement and its

presence within the BCR coreceptor complex enables a component of the
innate immune response (complement) to synergise with the BCR to

productively activate B-cells. Imagine a bacterium that has activated
complement and has become coated with the products of complement
activation, including C3d. If the same bacterium is subsequently captured
by the BCR on a B-cell, there is now an opportunity for CR2 within the

BCR coreceptor complex to bind C3d, which effectively means that the B-
cell now receives two signals simultaneously. Signal one comes via the
BCR and signal two via the coreceptor complex. So how does simultaneous
engagement of the coreceptor complex and the BCR lead to enhanced B-

cell activation?
Well, the answer is that we don’t know for sure, but it is clear that CD19
plays an especially important role in this process. CD19 is a B-cell-specific
transmembrane protein that is expressed from the pro-B-cell to the plasma-

cell stage and possesses a relatively long cytoplasmic tail containing nine
tyrosine residues. Upon B-cell receptor stimulation, the cyto-plasmic tail of
CD19 undergoes phosphorylation at several of these tyrosine residues (by

kinases associated with the BCR) that creates binding sites on CD19 for
several proteins, including the tyrosine kinase Lyn, Vav and
phosphatidylinositol 3-kinase (PI3K). CD19 plays a role as a platform for
recruitment of several proteins to the BCR complex (Figure 8.18), much in
the same way that LAT functions in T-cell receptor activation.

Vav is recruited to CD19 upon phosphorylation of the latter by Lyn and,
along with PI3K that is also recruited to CD19 as a result of Lyn-mediated

phosphorylation (Figure 8.18), plays a role in the activation of the
serine/threonine kinase Akt; the latter may also enhance NFAT activation
through neutralizing the inhibitory effects of GSK3 (glycogen synthase
kinase 3) on NFAT Because GSK3 can also phos-phorylate and destabilize
Myc and cyclin D, which are essential for cell cycle entry, Akt activation

also has positive effects on proliferation of activated B-cells.
Similar to the role that CD28 plays on T-cells, the B-cell coreceptor
amplifies signals transmitted through the BCR approximately 100-fold. As

we have discussed above, because CD19 and CR2 (CD21) molecules enjoy
mutual association, this can be brought about by bridging the lg and CR2
receptors on the B-cell surface by antigen-C3d complexes bound to the

surface of APCs. Thus, antigen-i nduced clustering of the B-cell coreceptor
complex with the BCR lowers the threshold for B-cell activation by

bringing kinases that are associated with the BCR into close proximity with
the coreceptor complex. The action of these kinases on the coreceptor
complex engages signaling pathways that reinforce signals originating from
the BCR.



B-cells also require co-stimulation from T-helper

cells


Just as T-cells require co-stimulatory signals from DCs in the form of B7
ligands for productive activation (Figure 8.3), T-dependent B-cells also
require costimulation from T-helper cells in order to cross the threshold
required for clonal expansion and differentiation to effector cells (Figure
8.15). The sequence of events goes much like this. Upon encountering

cognate antigen through direct binding to a microorganism, the BCR
undergoes the initial activation events described above. This culminates in
the internalization of the BCR, along with captured antigen, which is then

processed and presented on MHC class II molecules (Figure 8.20). To
continue the process of maturation to either a plasma cell or a memory cell,
the B-cell must now encounter a T-cell capable of recognizing one of the
antigenic peptides the B-cell is now presenting from the antigen it has
internalized. Note that this need not be the same epitope recognized by the

B-cell to undergo initial activation. Upon encountering a T-cell with the
appropriate TCR, the B-cell provides stimulation to the T-cell in the form of
MHC–peptide as well as co-s timulatory B7 signals (Figure 8.20). In turn,

the T-cell upregulates CD40 ligand (CD40L) that can provide essential co-
stimulation to the B-cell, enabling the latter to become fully activated and
undergo clonal expansion and class switching. If CD40L help is not
forthcoming, B-cells rapidly undergo apoptosis and are eliminated. Thus, B-
cells and T-cells provide mutual co-stimulation as a means of reinforcing

their initial activation signals (Figure 8.20).

Figure 8.20. CD40-CD40L-dependent B-cell co-stimulation by a T-
helper cell.
Independently activated T-and B-cells can interact if the B-cell is presenting

the correct peptide-MHC complex sufficient for stimulation of the T-cell.
Successful antigen presentation by a B-cell to an activated T-helper cell

results in CD40L-dependent co-stimulation of the B-cell as well as the
provision of cytokines, such as IL-4, by the T-cell that are essential for class
switching, clonal expansion and differentiation to effector cells.





































In effect, the B-lymphocyte is acting as an antigen-presenting cell and, as
mentioned above, it is very efficient because of its ability to concentrate the

antigen by focusing onto its surface Ig. Nonetheless, although a
preactivated T-helper can mutually interact with and stimulate a resting B-
cell, a resting T-cell can only be triggered by a B-cell that has acquired the
B7 co-s timulator and this is only present on activated, not resting, B-cells.

Presumably the immune complexes on follicular dendritic cells in
germinal centers of secondary follicles can be taken up by the B-cells for
presentation to T-helpers, but, additionally, the complexes could cross-link

the sIg of the B-cell blasts and drive their proliferation in a T-independent
manner. This would be enhanced by the presence of C3 in the complexes as
the B-cell complement receptor (CR2) is comitogenic.


Damping down B-cell activation

We have already discussed how T-cell enthusiasm for antigen can be
dissipated by engaging CTLA-4; similar mechanisms also operate to damp

down signals routed through the BCR. Several cell surface receptors,
including FcyRIIB, CD22 and PIRB (paired immunoglobulin-like receptor
B), have been implicated in antagonizing B-cell activation through
recruitment of the protein tyrosine phosphatase SHP-1 to ITIMs

(immunoreceptor tyrosine-based inhibitory motifs) in their cytoplasmic
tails. SHP-1 impairs BCR signaling by antagonizing the effects of the Lyn
kinase on Syk and Btk; by dephos-phorylating both of these proteins SHP-1
blocks recruitment of PLCγ2 to the BCR complex. Coligation of the BCR

with any of these receptors is therefore likely to block B-cell activation.
CD22 appears to be constitutively associated with the BCR in resting B-
cells and in this way may raise the threshold for B-cell activation.
Successful formation of a B-cell receptor synapse may physically exclude

CD22 from the BCR complex.



Dynamic interactions at the BCR synapse


Just as TCRs form immunological synapses during contact with specific

peptide—MHC, B-cell receptors have also been found to exhibit similar
behavior, particularly when antigen is presented on a membrane surface.
While B-cells can be stimulated by soluble antigen, it is now widely
accepted that the primary form of antigen that triggers B-cell activation in

vivo is localized to membrane surfaces. The most likely culprits here are the
follicular dendritic cells that are resident within lymph nodes, as well as
macrophages and DCs that migrate there bearing gifts of antigen. Antigens
can be immobilized on cell surfaces by complement or Fc receptors as

immuno-complexes, or through direct binding to various scavenger
receptors. An encounter between a B-cell and membrane-associated antigen
provides the opportunity for the B-cell membrane to spread along the
opposing membrane, gathering sufficient antigen to trigger B-cell

activation, as well as providing an opportunity for other contacts to be made
such as those that can be provided by membrane integrins. This spreading
response is driven by BCR engagement of antigen at the leading edge of the
B-cell and, apart from increasing the number of BCR-antigen contacts that

are then available to trigger B-cell activation, the spreading response also
increases the amount of antigen that is ultimately concentrated and

internalized by the B-cell-leading to more efficient antigen presentation to
activated T-cells when the B-cell subsequently goes looking for T-cell help
(Figure 8.20).

Figure 8.21. The B-cell receptor (BCR) immunological synapse.

(a) Imaging of the BCR immunological synapse. Real-time quantification
of antigen and ICAM-1 recruitment to the B-cell synapse. naive B-cells
were settled onto planar lipid bilayers containing
glycosylphosphatidylinositol (GPI)-linked ICAM-1 (red) and p31 antigen

(green). Central panels show the accumulation of the antigen p31 (green)
and ICAM-1 (red) in the pattern of a mature synapse at the specified time
points. Top and bottom panels show differential interference contrast and
interference reflection microscopy images of the same time points.

(Reproduced with permission from Carrasco Y.R., Fleire S.J., Cameron T.,
Dustin M.L. & Batista F.D. (2004) Immunity 20, 589–599, © Elsevier). (b)
Schematic representation of the BCR immunological synapse, depicting the
central supramolecular activation complex (cSMAC) that is enriched in

BCR-Ag microclusters, and the surrounding peripheral supramolecular
activation complex (pSMAC) that is enriched in integrins such as LFA-
1/ICAM-1.

Cell spreading in response to engagement of the BCR with specific
antigen is triggered in response to signals propagated via the BCR, with Lyn
and Syk playing especially important roles in this process. Clearly,
spreading along an antigen-bearing surface requires extensive
reorganization of the cytoskeleton. Although this is not fully understood at

present, activation of Vav, which as discussed earlier is involved in the
regulation of the cytoskeleton via Rac and Rho, is essential here.
There is evidence that BCRs within resting B-cells are not scattered

randomly within the plasma membrane but are confined to certain zones,
with free diffusion restricted by contacts with the underlying actin-based
cytoskeleton. In line with this, disruption of the actin network in B-cells has
been shown to lead to spontaneous BCR-dependent calcium signaling,

possibly due to the spontaneous formation of BCR microclusters. Thus, the
cytoskeleton appears to play an important role in restricting the surface

distribution and behavior of BCRs in a resting B-cell. Binding of
multivalent antigen to the BCR can disrupt the arrangement of BCRs in the

resting B-cell resulting in the formation of BCR microclusters containing
50500 BCRs, the formation of that also depends on an intact cytoskeleton.
Indeed, the actin network within activated B-cells has been found to
encircle or corral BCR microclusters within the plasma membrane.

Spreading of the B-cell across the antigen-bearing surface increases the
number of BCR microclusters and eventually engages sufficient numbers of
BCRs to permit crossing of the threshold for B-cell activation. Similar to T-

cells, mature B-cells also express high levels of the LFA-1 and VLA-4
integrins. Interaction of these adhesion molecules with their cognate
ligands, ICAM-1 and VCAM-1/fibronectin, on the cell that is displaying the
immobilized antigen also promote B-cell adhesion and facilitate cell
spreading along the target surface. Following spreading across an antigen-

bearing surface, B-cells undergo a prolonged contraction phase that
culminates in a major rearrangement of the BCR microclusters within the
membrane that coalesce to form an immunological synapse, similar to that

seen with T-cells (Figure 8.21). The mature BCR immunological synapse
contains a central ring (cSMAC) enriched in BCR-antigen complexes, with
an outer ring (pSMAC) enriched in integrins (Figure 8.21). No only do the
integrin contacts promote spreading and adhesion between the interacting
cell pairs, but recent evidence also suggests that such contacts lower the

threshold for B-cell activation by lowering the concentration of antigen
required to form a stable synapse and trigger the B-cell.


SUMMARY



Immunocompetent T-and B-cells differ in many respects
The antigen-specific receptors, TCR/CD3 on T-cells and surface lg
on B-cells, provide a clear distinction between these two cell types.

T-and B-cells differ in their receptors for C3d, IgG and certain
viruses.
There are distinct polyclonal activators of T-cells (PHA, anti-CD3)

and of B-cells (anti-Ig, Epstein–Barr virus).

T-lymphocytes and antigen-presenting cells interact through pairs
of accessory molecules

The docking of T-cells and APCs depends upon strong mutual
interactions between complementary molecular pairs on their surfaces:
MHC II–CD4, MHC I–CD8, VCAM–1–VLA–4, ICAM–1–LFA–1,
LFA–3–CD2, B7–CD28 (and CTLA–4).

B7-CTLA-4 interactions are inhibitory, whereas B7–CD28
interactions are stimulatory. CTLA-4 may antagonize the recruitment

of the TCR to lipid rafts where many membrane-associated signaling
proteins reside.
Activation of T-cells requires two signals

Two signals activate T-cells, but one alone produces
unresponsiveness (anergy) or death via apoptosis.
Signal 1 is provided by the low affinity cognate TCR–MHC plus

peptide interaction.
The second co-stimulatory signal (signal 2) is mediated through
ligation of CD28 by B7 (CD80/ CD86) and greatly amplifies signals
generated through TCR-MHC interactions.

Previously stimulated T-cells require only one signal, through their
TCRs, for efficient activation.

T-cell receptor activation
The TCR does not possess any intrinsic enzymatic activity but is
associated with accessory proteins (the CD3 coreceptor complex) that
can recruit protein tyrosine kinases (PTKs).

The TCR signal is transduced and amplified through a protein
tyrosine kinase enzymic cascade.

Recruitment of CD4 or CD8 to the TCR complex leads to
phosphorylation of ITAM sequences on CD3-associated ζ chains by
the CD4-associated Lck PTK. The phosphorylated ITAMs bind and
then activate the ZAP-70 kinase.

Downstream events following TCR signaling
Nonenzymic adaptor proteins form multimeric complexes with

kinases and guanine nucleotide exchange factors (GEFs).

Hydrolysis of phosphatidylinositol diphosphate by phospholipase
Cγ1 or Cγ2 produces inositol triphosphate (IP ) and diacylglycerol
3
(DAG).
IP mobilizes intracellular calcium.
3
DAG and increased calcium activate protein kinase C.

The raised calcium together with calmodulin also stimulates
calcineurin activity.
Activation of Ras by the guanine nucleotide exchange factor Sos sets

off a kinase cascade operating through Raf, the MAP kinase kinase
MEK and the MAP kinase ERK. CD28 through PI3 kinase can also
influence MAP kinase.

The transcription factors Fos and Jun, NFAT and NFκB are activated
by MAP kinase, calcineurin and PKC, respectively, and bind to
regulatory sites in the IL-2 promoter region.

A small number of MHC–peptide complexes can serially trigger a
much larger number of TCRs thereby providing the sustained signal
required for activation.

Initial binding of integrins facilitates the formation of an
immunological synapse, the core of that exchanges integrins for TCR
interacting with MHC–peptide.
Cbl family adaptor molecules are involved in negative signaling

pathways.
The phosphatase domains on CD45 are required to remove

phosphates at inhibitory sites on kinases.
B-cells respond to three different types of antigen
Type 1 thymus-independent antigens are polyclonal activators

focused onto the specific B-cells by sIg receptors.
Type 2 thymus-independent antigens are polymeric molecules that
cross-link many sIg receptors and, because of their long half-lives,

provide a persistent signal to the B-cell.
Thymus-dependent antigens require the cooperation of helper T-cells
to stimulate antibody production by B-cells.

Antigen captured by specific sIg receptors is taken into the B-cell,
processed and expressed on the surface as a peptide in association with

MHC II.
This complex is recognized by the T-helper cell that activates the
resting B-cell.

The ability of protein carriers to enable the antibody response to
haptens is explained by T-cell–B-cell collaboration, with T-cells
recognizing the carrier and B-cells the hapten.

The nature of B-cell activation
Cross-linking of surface lg receptors (e.g. by type 2 thymus-
independent antigens) activates B-cells.

T-helper cells activate resting B-cells through TCR recognition of
MHC II–carrier peptide complexes and co-stimulation through
CD40L-CD40 interactions (analogous to the B7-CD28 second signal

for T-cell activation).
B-cell co-stimulation is also provided by the B-cell coreceptor
complex consisting of CD19, CD21, CD81 and LEU13.

B-cell receptors (BCRs) also form immunological synapses
composed of numerous BCR microclusters and integrins.


FURTHER READING



Abraham R.T. & Weiss A. (2004) Jurkat T-cells and development of the T-
cell receptor signaling paradigm. Nature Reviews Immunology 4, 301–308.
Acuto O. & Michel F. (2003) CD28-mediated costimulation: a quantitative

support for TCR signaling. Nature Reviews Immunology 3, 939–951.

Batista F.D. & Harwood N.E. (2009) The who, how and where of antigen
presentation to B cells. Nature Reviews Immunology 9, 15–27.
Buday L. & Downward J. (2008) Many faces of Ras activation. Biochim

Biophys Acta. 1786, 178–187.
Bromley S.K. et al. (2001) The immunological synapse. Annual Review of

Immunology 19, 375–396.
Fooksman D.R. et al. (2010) Functional anatomy of T cell activation and

synapse formation. Annual Review of Immunology 28, 1–27.

Grakoui A. et al. (1999) The immunological synapse: a molecular machine
controlling T-cell activation. Science 285, 221–227.

Harwood N.E. & Batista F.D. (2010) Early events in B cell activation.
Annual Review of Immunology 28, 185–210.

Huang F. & Gu H. ( 2008 ) Negative regulation of lymphocyte development
and function by the Cbl family of proteins. Immunological Reviews 224,

229–238.
Jenkins M.K. et al. (2001) In vivo activation ofantigen-specific CD4 T-cells.
Annual Review ofImmunology 19, 23–45.

Kinashi T. (2005) Intracellular signaling controlling integrin activation in

lymphocytes. Nature Reviews Immunology 5, 546–559.
Kurosaki T. (2002) Regulation of B-cell signal transduction by adaptor
proteins. Nature Reviews Immunology 2, 354–363.

Mueller D.L. (2010) Mechanisms maintaining peripheral tolerance.
NatureImmunology 11, 21–27.

Niiro H. & Clark E.A. (2002) Regulation of B-cell fate by antigen-receptor
signals. Nature Reviews Immunology 2, 945–956.

Rudd C.E., Taylor A. & Schneider H. (2009) CD28 and CTLA-4 coreceptor
expression and signal transduction. Immunological Reviews 229, 12–26.

Smith-Garvin J.E., Koretzky G.A. & Jordan M.S. (2009) T cell activation.
Annual Review of Immunology 27, 591–619.

Yokosuka T. & Saito T. (2009) Dynamic regulation of T-cell costimulation
through TCR-CD28 microclusters. Immunological Reviews 229, 27–40.




Now visit www.roitt.com to test yourself on this chapter.

CHAPTER 9

The production of effectors






Key Topics

Effector mechanisms

Cytokines act as intercellular messengers
Activated T-cells proliferate in response to cytokines

Different T-cell subsets can make different cytokine patterns
Cells of the innate immune system shape the Th1/Th2/Th17 response

Policing the adaptive immune system
+
CD8 T-cell effectors in cell-mediated immunity
Proliferation and maturation of B-cell responses are mediated by cytokines

What is going on in the germinal center?
The synthesis of antibody

Immunoglobulin class switching occurs in individual B-cells
Factors affecting antibody affinity in the immune response
Memory cells



Just to Recap. ..



The reader will be familiar with the central role of mature dendritic cells
(DCs) as sources of MHC-peptide (signal 1) and co-stimulatory ligands
(signal 2) for productive activation of naive T-cells; the absence of signal 2
leads to unresponsiveness or anergy. B-cells can also present antigen and
co-stimulate a previously activated T-cell, for the purposes of receiving T-

cell help for clonal expansion and differentiation to an effector B-cell, but
DCs are typically the primary antigen-presenting cells (APCs) for activation
of naive T-cells. Macrophages can also act as APCs but, due to their

relatively nonmigratory behavior, have a greater role in restimulating
previously activated T-cells at sites of infection rather than priming naive T-
cells within secondary lymphoid tissues. The goal of antigen-driven

lymphocyte activationis to trigger clonal expansion of the correct cells so
that these cells can set to work mounting an adaptive immune response. The

adaptive immune response works in tandem with an ongoing innate
immune response and, as we shall see, amplifies and reinforces the innate
immune response through the provision of cytokines, antibodies, and
cytotoxic molecules. Activated T-cells differentiate into effector cells

capable of secreting diverse patterns of cytokines; similarly, activated B-
cells differentiate into plasma cells capable of secreting different antibody
classes. Cells of the innate immune system in general, and DCs in
particular, play a key role in shaping the particular flavor of adaptive

immune response that is mounted in response to antigenic stimulation. This
is achieved through the secretion of different patterns of cytokines in
response to the initial infection. These cytokines, in turn, influence the
nature of the T-and B-cell effectors that are produced.



Introduction



Having crossed the threshold required for activation, a T-cell enters the cell
division cycle and undergoes clonal proliferation and differentiation to
effectors. A succession of genes is upregulated upon T-cell activation.
Within the first 30 minutes, nuclear transcription factors such as Fos/Jun
and NFAT, which regulate interleukin-2 (IL-2) expression and the cellular

proto-oncogene c-myc, are expressed, but the next few hours see the
synthesis of a range of cytokines and their specific receptors. Much later we
see molecules like the transferrin receptor related to cell division and very

late antigens such as the adhesion molecule VLA-1 that enables activated T-
cells to bind to vascular endothelium at sites of infection. Collectively, these
events equip activated T-cells with new functional properties, which include
the ability to activate macrophages, provision of cytokine-mediated help for
antibody production by B-cells, and the ability to eliminate virally infected

targets by inducing apoptosis in such cells.
Activated B-cells also enter the cell cycle and undergo clonal
proliferation to swell their ranks. Some of the activated B-cells eventually

differentiate into plasma cells that migrate to the bone marrow where they
produce and secrete large amounts of antibody for relatively long periods.

Here we will consider the main issues surrounding the acquisition of
effector function by T-and B-lymphocytes and the roles that effector

lymphocytes play in the immune response.



Effector mechanisms




The innate immune system utilizes a number of

different effector mechanisms to combat infection


In Chapter 1 we learned that the innate immune system uses a variety of
strategies to deal with microorganisms that have successfully breached the
physical barriers of the skin and mucosal surfaces. The first line of defense
involves the cells and soluble factors of the innate immune system that take

immediate action upon detection of nonself in the form of pathogen-
associated molecular patterns (PAMPs). The steps taken by the innate
immune system in dealing with a nascent infection range from direct

binding of soluble pattern recognition molecules—t uch as complement,
lysozyme and man-nose-binding lectin—to orchestrate immediate
destruction of a pathogen, or to enhance phagocytic uptake by macrophages
and neutrophils. Macrophages and neutrophils also directly recognize and
engulf pathogens via their cell associated pattern recognition receptors.

Other options at the disposal of the innate immune system involve the
deployment of mast cells and basophils, both of which use their granule
enzymes to combat large extracellular parasites. Let us also not forget

natural killer (NK) cells that are adept at killing host cells displaying signs
of viral infection or other evidence of trouble within. We also discussed the
role of dendritic cells as sentinels of the innate immune system, serving to
alert T-cells to an ongoing infection by presenting antigen within the
context of appropriate coittimulatory signals (i.e. ligands of the B7 family).

All told, there are a number of highly effective weapons in the arsenal of
the innate immune system that can be called into play for the purposes of
defending the body against infection. Nonetheless, the innate immune

system frequently requires assistance to deal with well adapted pathogens
that deploy a range of immune evasion strategies to frustrate all of the

above efforts. The cavalry comes in the form of the adaptive immune
response.



Adaptive immunity also employs a range of

effector mechanisms

The adaptive immune system, made up ofT-and B-lymphocytes, also has a

number of weapons at its disposal. In Chapter 2 we discussed the role of B-
cell-derived antibodies as a means of coating microorganisms for the
purposes of enhancing complement-mediated lysis via the classical

pathway, or enhancing their uptake by phagocytosis via specific Fc
receptors on macrophages and neutrophils, or indeed by simply aggregating
infectious agents and impeding their further incursion into tissues.
Antibodies can also be used to the advantage of NK cells to focus their
cytotoxic actions via antibody-dependent cellular cytotoxicity (ADCC) (see

p. 48). T-cells also employ a number of different strategies to defend the
body from infectious agents (Figure 9.1). Recall that T-cells can be grouped
into two major subdivisions: helper (Th) and cytotoxic (Tc or CTL) T-cells,

that are selected to recognize antigen presented in the context of MHC class
II or MHC class I molecules, respectively. Whereas T-helper cells function
to help B-cells make antibodies or to activate the killing function of
macrophages or NK cells, Tc cells are endowed with the ability to engage
and kill virally infected cells. As we shall discuss in more detail in this

chapter, T-helper cells can be further subdivided into Th1, Th2 and
Th17 cells on the basis of the cytokine profiles that these cells secrete, as
this confers different effector functions on such cells. We shall also discuss

other subdivisions of T-cells (regulatory T-cells [Tregs ]t, that also differ in
the cytokine profile produced, as these cells serve important regulatory
functions and help to safeguard the body against inappropriate T-cell
responses that are directed against self, as well as excessive or inappropriate
responses directed towards nonself.



Cytokines heavily influence the generation as well

as the specific function of effectors within the

adaptive immune system


The production of various cytokines is central to both the maturation as
well as the specific effector functions of B-and T-cells. We have already
referred to the diverse roles of cytokines throughout the previous chapters
and have alluded to their properties as messenger molecules that enable the

disparate elements of the immune system to communicate with each other.
Communication between cells of the immune system underpins the
amplification of immune responses (Figure 9.1), and is also instrumental in
marshalling the appropriate response (i.e. whether predominantly antibody

mediated or cell mediated) depending on the nature of the infectious agent
as well as its route of entry into the body. Here, we will go into more detail
concerning the different categories of cytokines, how these molecules act
upon their target cells, and the spectrum of responses they initiate. All of

these issues are central to how the effector cells of the adaptive immune
system are generated and the nature of the responses they engage in.
Whereas many of the elements of the innate immune system are poised to
strike with little delay upon detection of a PAMP, the actions of T-and B-

lymphocytes are heavily influenced by the cytokine environment
accompanying their initial exposure to specific antigen.


Dendritic cells and other cells of the innate

immune system play a central role in the


generation of effectors

As we shall discuss later in this chapter, a major influence on the type of
effector cells generated in response to a pathogenic challenge is wielded by

dendritic cells that—in addition to presenting antigen (signal 1) and
providing co-stimulatory signals (signal 2) to T-cells—also exert significant
control over the type of T-cell response that is generated. Dendritic cells
achieve this by providing additional input in the form of cytokines (signal

3) that shape the nature of the effector T-cells that are thus generated
(Figure 9.2). The particular cocktail of cytokines elaborated by DCs during
the initial round of T-cell stimulation in a lymph node influences whether
the response will be dominated by the generation of T-cell effectors that

provide help for Bt cells (Th2), or alternatively, result in the generation of

T-cells that activate macrophages and assist CTL function (Th1 cells). The
generation of other Th subsets, characterized by particular patterns of

cytokines, has also been recognized. The pattern of cytokines secreted by
differentiated effector T-cells can be further influenced by local
macrophages, NK cells, basophils and other cells of the innate immune
system encountered by activated T-cells that migrate to sites of infection.

Once again, this is through the provision of cytokines that trigger or
reinforce the development of different T-cell effector subsets. This
inevitably raises the question of what influences DCs to make one pattern
of cytokines over another. The answer to this neatly brings us full circle, as

it is the nature of the PAMPs that propel DCs into action in the first place,
as well as cytokines elaborated by the other cells of the innate immune
system upon encountering an infectious agent, that influence the cytokine
profile adopted by an activated DC. Before we discuss the various T-and B-

lymphocyte effector cell types, let us first take a closer look at the diversity
of the cytokine family and how these important cell-cell communication
molecules exert their effects at a molecular level.

Figure 9.1. T-cells can regulate diverse elements of the immune system

through the production of different cytokines.
This illustrates some, but by no means all, of the interactions that activated
T-cells can have with other elements of the immune system through the
directed secretion of specific cytokines. Note that not all T-cells are capable

of secreting all of the cytokines indicated. Rather, specific T-cell subsets are
generated that are skewed towards secretion of particular subsets of the
cytokines shown.

Figure 9.2. Generation of effector T-cells is influenced by the cytokine

environment experienced by the T-cell at the point of initial activation.
MHC-peptide recognition by the TCR represents signal 1, co-stimulation of
CD28 by B7 ligands represents signal 2, and cytokines produced by the DC

represents signal 3. Note that the cytokine environment upon restimulation
of a T-cell within an infected tissue will also influence the nature of the
effector response made by the T-cell.

Cytokines act as i ntercellular messengers






Cytokines are structurally diverse polypeptides that function as messenger
molecules that can communicate signals from one cell type to another and,
amongst other things, can instruct the cell receiving the signal to proliferate,

differentiate, secrete additional cytokines, migrate or die. To date, many
different cytokines have been described and no doubt some remain to be
discovered (Table 9.1). One of the most important cytokine groupings, to
the immunologists way of thinking, is the interleukin family as this contains

cytokines that act as communicators between leukocytes. Members of the
interleukin family are very diverse, belonging to different protein structural
classes (Figure 9.3), because the primary qualification for membership of
this family is biological (i.e. evidence of activity on leukocytes) rather than

sequence or structural homology. Indeed, while additional homologs of the
interleukin family are known, their status as interleukins awaits evidence
that these proteins exert functional effects upon leukocytes. Approximately
34 interleukins have been described to date (IL-1 to IL-35) with the status

of IL-14 as an interleukin in doubt.
Other cytokine families have been established on the basis of their ability
to support proliferation of hematopoietic precursors (colony stimulating

factors), or cytotoxic activity towards transformed cell types (tumor
necrosis factors), or the ability to interfere with viral replication
(interferons). It is important to note, however, that cytokines frequently
have pleiotrophic effects, doing much more than their somewhat descriptive

(and often misleading) names would suggest. Indeed, the response that
many of these molecules elicit depends, to a large extent, on the context in
which the cytokine signal is delivered. Thus, factors such as the
differentiation stage of the cell, its position within the cell cycle (whether

quiescent or proliferating) and the presence of other cytokines, can all
influence the response made to a particular cytokine.

Table 9.1 Cytokines: their origin and function. APR acute phase
proteins; B, B-cell; baso, basophil; BM, bone marrow; Endo, endothelium;

eosino, eosinophil; Epith, epithelium; Fibro, fibroblast; GM-CSF,
granulocyte-macrophage colony-stimulating factor; IL, interleukin; LIF,

leukemia inhibitory factor; macrophage; MC, mast cell; Mono, monocyte;
neutro, neutrophil; NK, natural killer; SLF, steel locus factor; T, T-cell;

TGFß, transforming growth factor-ß. Note that there is not an interleukin-
14. This designation was given to an activity that, upon further
investigation, could not be unambiguously assigned to a single cytokine.
IL-30 also awaits assignment. IL-8 is a member of the chemokine family.

These cytokines are listed separately in Table 9.2.
Cytokine Source Effector function

Interleukins
IL-1 α, IL-1 Mono, Mϕ Co-stimulates T activation by enhancing production of cytokines
β DC, NK, B, including IL-2 and its receptor; enhances B proliferation and
Endo maturation; NK cytotoxicity; induces IL-1,-6,-8, TNF, GM-CSF and
PGE by Mϕ; proinflammatory by inducing chemokines and ICAM-
2
1 and VCAM-1 on endothelium; induces fever, APP bone resorption
by osteoclasts
IL-2 Th1 Induces proliferation of activated T-and B-cells; enhances NK
cytotoxicity and killing of tumor cells and bacteria by monocytes and
Mϕ

IL-3 T, NK, MC Growth and differentiation of hematopoietic precursors; MC growth
IL-4 Th2, Tc2, Induces Th2 cells; stimulates proliferation of activated B, T, MC;
NK, NKT, γδ upregulates MHC class II on B and and Mϕ and CD23 on B;
T, MC downregulates IL-12 production and thereby inhibits Th1
differentiation; increases phagocytosis; induces switch to IgG1 and
IgE
IL-5 Th2, MC Induces proliferation of eosinophils and activated B; induces switch to
IgA
IL-6 Th2, Mono, Differentiation of myeloid stem cells and of B into plasma cells;
Mϕ, DC, BM induces APP; enhances T proliferation
stroma
IL-7 BM and Induces differentiation of lymphoid stem cells into progenitor T and
thymic B; activates mature T
stroma
IL-8 Mono, Mϕ, Mediates chemotaxis and activation of neutrophils
Endo
IL-9 Th Induces proliferation of thymocytes; enhances MC growth; synergizes
with IL-4 in switch to IgG1 and IgE
IL-10 Th (Th2 in Inhibits IFNγ secretion by mouse, and IL-2 by human, Th1 cells;
mouse), Tc, downregulates MHC class II and cytokine (including IL-12)
B, Mono, production by mono, Mϕ, and DC, thereby inhibiting Th1
Mϕ differentiation; inhibits T proliferation; enhances B differentiation

IL-11 BM stroma Promotes differentiation of pro-B and megakaryocytes; induces APP
IL-12 Mono, Mϕ, Critical cytokine for Th1 differentiation; induces proliferation and
DC, B IFNγ production by Th1, CD8 and γδ T and NK; enhances NK and
+
+
CD8 T cytotoxicity
IL-13 Th2, MC Inhibits activation and cytokine secretion by Mϕ; co-activates B
proliferation; upregulates MHC class II and CD23 on B and mono;
induces switch to IgG1 and IgE; induces VCAM-1 on endo
IL-15 T, NK, Induces proliferation of T-, NK and activated B and cytokine
Mono, Mϕ, production and cytotoxicity in NK and CD8 T-cell; chemotactic for
+
DC, B T-cell; stimulates growth of intestinal epithelium
IL-16 Th, Tc Chemoattractant for CD4 T, mono and eosino; induces MHC class II

IL-17 T Proinflammatory; stimulates production of cytokines including
TNF,IL-1β,-6,-8, G-CSF

IL-17A Th17, T-cells Proinflammatory, stimulates production of cytokines including TNF,
NK, IL-1β, IL-6, -8, G-CSF by epithelia, endothelia and fibroblasts.
Neutrophils
IL-17F Th17, T-cells Similar effects to IL-17A
NK,
Neutrophils
IL-18 Mϕ, DC Induces IFNy production by T; enhances NK cytotoxicity
IL-19 Mono Modulation of Th1 activity

IL-20 Mono, Regulation of inflammatory responses to skin
Keratinocytes

IL-21 Th Regulation of hematopoiesis; NK differentiation; B activation; T co-
stimulation

IL-22 T Inhibits IL-4 production by Th2
IL-23 DC Proliferation and IFNγ production by Th1, induces expansion and
survival of TH17 cells. induction of proinflammatory cytokines such
as IL-1, IL-6, TNF by macrophages.

IL-24 Th2, Mono, Induction of TNF, IL-1, IL-6, anti-tumor activity
Mϕ

IL-25 Th1, Mϕ, Induction of IL-4, IL-5, IL-13 and Th2-associated pathologies
Mast
IL-26 T, NK Enhanced production of IL-8 and IL-10 by epithelium
IL-27 DC, Mono Induction of TH1 responses, enhanced IFN-γ production

IL-28 Mono, DC Type 1 IFN-like activity, inhibition of viral replication
IL-29 Mono, DC Type 1 IFN-like activity, inhibition of viral replication
IL-30 APCs P28 subunit of IL-27 heterodimer. Regulates 1L-12 responsiveness of
naive T-cells. Synergizes with IL-12 to induce IFN-γ.

IL-31 T Promotes inflammatory responses in skin

IL-32 NK, T Promotes inflammation. Role in activation-induced T-cell apoptosis.
IL-33 Stroma, DC Induction of Th2 cytokines, mediates chemotaxis of basophils and
mast cells
IL-34 Stroma Stimulates monocyte proliferation and formation of macrophage
progenitors.
IL-35 Tregs Immunosuppressive effects on Th1, Th2 and Th17 cells. Stimulates
proliferation of Tregs.
Colony
stimulating
factors
GM-CSF Th,Mϕ Stimulates growth of progenitors of mono-, neutro-, eosino-and
Fibro, MC, basophils; activates Mϕ
Endo

G-CSF Fibro, Endo Stimulates growth of neutro progenitors
M-CSF Fibro, Endo, Stimulates growth of mono progenitors
Epith
SLF BM stroma Stimulates stem cell division (c-kit ligand)
Tumor
necrosis
factors
TNF (TNFα) Th, Mono, Tumor cytotoxicity; cachexia (weight loss); induces cytokine
Mϕ DC, MC, secretion; induces E-selectin on endo; activates Mϕ antiviral
NK, B

Lymphotoxin Th1, Tc Tumor cytotoxicity; enhances phagocytosis by neutro and Mϕ
(TNFβ) involved in lymphoid organ development; antiviral

Interferons
IFNα Leukocytes Inhibits viral replication; enhances MHC class II
IFNβ Fibroblasts Inhibits viral replication; enhances MHC class II

IFNγ Th1, Tc1, NK Inhibits viral replication; enhances MHC class I and II; activates Mϕ
induces switch to IgG2a; antagonizes several IL-4 actions; inhibits
proliferation of Th2
Others

TGFβ Th3, B, Mϕ Proinflammatory by, e.g. chemoattraction of mono and Mϕ but also
MC antiinflammatory by, e.g. inhibiting lymphocyte proliferation; induces
switch to IgA; promotes tissue repair
LIF Thymic Induces APP
epith, BM
stroma
Eta-1 T Stimulates IL-12 production and inhibits IL-10 production by Mϕ

Oncostatin T, Mϕ Induces APP
M



Cytokine action is transient and usually short

range

Cytokines are typically low-molecular-weight (15–25kDa) secreted proteins

that mediate cell division, inflammation, cytotoxicity, differentiation,
migration and repair. Because they regulate the amplitude and duration of
the immune– inflammatory responses, cytokines must be produced in a
transient manner tightly coupled to the presence of foreign material.

Cytokine production can also occur in response to the release of
endogenous “danger signals” (i.e. danger-associated molecular patterns
[DAMPs]) that betray the presence of cells dying by necrosis, a mode of
cell death that is typically seen in pathological situations and frequently

provoked by infectious agents or tissue injury (see Chapter 1, p. 4). It is
relevant that the AU-rich sequences in the 3’-untranslated regions of the
mRNA of many cytokines prime these mRNAs for rapid degradation
thereby ensuring that cytokine production rapidly declines in the absence of

appropriate stimulation. Unlike endocrine hormones, the majority of
cytokines normally act locally in a paracrine or even autocrine fashion.
Thus cytokines derived from lymphocytes rarely persist in the circulation,
but nonlymphoid cells such as endothelial cells and fibroblasts can be

triggered by bacterial products to release cytokines that may be detected in
the bloodstream, often to the detriment of the host. Septic shock, for
example, is a life-threatening condition that largely results from massive
overproduction of cytokines such as tumor necrosis factor (TNF) and IL-1

in response to bacterial infection and highlights the necessity to keep a tight
rein on cytokine production. Certain cytokines, including IL-1 and TNF,
also exist as membrane-anchored forms and can exert their stimulatory
effects without becoming soluble.


Figure 9.3. Cytokine structures.
Cytokines can be divided into a number of different structural groups.
Illustrated here are three of the main types of structure and some named
examples of each type: (a) four short (~15 amino acids) α-helices, (b) four

long (~25 amino acids) α-helices and (c) a β-sheet structure. (Reproduced
with permission from Michal G. (ed.) (1999) Biochemical Pathways: An

Atlas of Biochemistry and Molecular Biology. John Wiley & Sons, New
York.)























Cytokines act through cell surface receptors



Cytokines are highly potent, often acting at femtomolar (10 -15 M)
concentrations, combining with small numbers of high affinity cell surface
receptors to produce changes in the pattern of RNA and protein synthesis in
the cells they act upon. This is achieved through cytokine receptor-mediated
activation of signal transduction cascades that culminate in the activation of

transcription factors that direct the synthesis of new gene products, or
increases the level of existing ones, within the cell. The end result is a
change in the behavior or functionality of the cell as a result of these

gene expression changes. Cytokine receptors typically possess specific
protein– protein interaction domains or phosphorylation motifs within their
cytoplasmic tails to facilitate recruitment of appropriate adaptor proteins
upon receptor stimulation. A recurring theme in cytokine receptor
activation pathways is the ligand-induced dimert or trimerization of

receptor subunits; this facilitates signal propagation into the cell through the
interplay of the transiently associated receptor cytoplasmic tails. There are
six major cytokine receptor structural families (Figure 9.4).


Hematopoietin receptors

Thiese are the largest family, sometimes referred to simply as the cytokine
receptor superfamily, and are named after the first member of this family to

be defined—the hematopoietin receptor. These receptors generally consist
of one or two polypeptide chains responsible for cytokine binding and an
additional shared (common or “c”) chain involved in signal transduction.
The γc (CD 132) chain is used by the IL-2 receptor (Figure 9.4a) and IL-4,

IL-7, IL-9, IL-15 and IL-21 receptors, a βc (CDw131) chain by IL-3, IL-5
and granulocyte-macrophage colonytstimulating factor (GM-CSF)
receptors, and gp130 (CD130) shared chain by the IL-6, IL-11, IL-12,
oncostatin M, ciliary neurotrophic factor and leukemia inhibitory factor

(LIF) receptors.

Figure 9.4. Cytokine receptor families.
One example is shown for each family. (a) The hematopoietin receptors

operate through a common subunit (γc, βc or gp130, depending on the
subfamily) that transduces the signal to the interior of the cell. In essence,
binding of the cytokine to its receptor must initiate the signaling process by
mediating hetero-or homodimer formation involving the common subunit.

In some cases the cytokine is active when bound to the receptor either in
soluble or membrane-bound form (e.g. IL-6). The IL-2 receptor is
interesting with respect to its ligand binding. The a chain (CD25, reacting
with the Tac monoclonal) of the receptor possesses two complement control

protein structural domains and binds IL-2 with a low affinity; the β chain
(CD122) has a membrane proximal fibronectin type III structural domain
and a membrane distal cytokine receptor structural domain, and associates
with the common γ chain (CD132) that has a similar structural organization.

The β chain binds IL-2 with intermediate affinity. IL-2 binds to and
dissociates from the a chain very rapidly but the same processes involving
the p chain occur at two or three orders of magnitude more slowly. When
the α, β and γ chains form a single receptor, the β chain binds the IL-2

rapidly and facilitates its binding to a separate site on the p chain from
which it can only dissociate slowly. As the final affinity (K ) is based on
d
-4 -
the ratio of dissociation to association rate constants, then K = 10 s
d
-1 -1
1 /10 M s = 10 -11 M, which is a very high affinity. The γ chain does
7
not itself bind IL-2 but contributes towards signal transduction. (b) The

interferon receptor family consists of heterodimeric molecules each of
which bears two fibronectin type III domains. (c) The receptors for TNF

and related molecules consist of a single polypeptide with four TNFR
domains. The receptor trimerizes upon ligand binding and, in common with
a number of other receptors, is also found in a soluble form that, when
released from a cell following activation, can act as an antagonist. (d)

Another group of receptors contains varying numbers of Ig superfamily
domains, whereas (e) chemokine receptors are members of the G-protein-
coupled receptor superfamily and have seven hydrophobic transmembrane
domains. (f) The final family illustrated are the TGF receptors that require

association between two molecules, referred to as TGFR type I and TGFR
type II, for signaling to occur.




























Interferon receptors

These also consist of two polypeptide chains and, in addition to the IFNα,

IFNβ and IFNγ) receptors (Figure 9.4b), this family includes the IL-10
receptor.


TNF receptors


Members of the TNF receptor superfamily possess cysteine-rich
extracellular domains and most likely exist as preformed trimers that
undergo a conformational change in their intracel-lular domains upon
ligand binding. They include the tumor necrosis factor (TNF) receptor

(Figure 9.4c) and the related Fas (CD95/APO-1) and TRAIL receptors. This
family also contains the lymphotoxin (LT) and nerve growth factor (NGF)

receptors, as well as the CD40 receptor, which plays an important role in
co-stimulation of B-cells and dendritic cells by activated T-cells.


IgSF cytokine receptors


Immunoglobulin superfamily members are broadly utilized in many aspects
of cell biology (cf. p. 309) and include the IL-1 receptor (Figure 9.4d), and
the macrophage colony-stimulating factor (M-CSF) and stem cell factor
(SCF/c-kit) receptors.



Chemokine receptors

Chemokines ( chemoattractant cyto kine) share a common functional
property of promoting chemotaxis and their receptors comprise a family of

approximately 20 different G-protein-coupled, seven transmembrane
segment polypeptides (Figure 9.4e). Each receptor subtype is capable of
binding multiple chemokines within the same family. For example, CXC
receptor 2 (CXCR2) is capable of binding seven different ligands within the

CXC ligand (CXCL) family.
The recruitment of T-cells, macrophages and neutrophils to an
inflammatory site is greatly enhanced by the action of chemokines. These

can be produced by a variety of cell types and are divided into four families
based on the disposition of the first (N terminal) two of the four canonical
cysteine residues (Table 9.2). CXC chemokines have one amino acid and
CX3C have three amino acids between the two cysteines. CC chemokines

have adjacent cysteines at this location, whereas C chemokines lack
cysteines 1 and 3 found in other chemokines. Chemokines bind to G-
protein-coupled seven transmembrane receptors (Figure 9.4). Despite the
fact that a single chemokine can sometimes bind to more than one receptor,

and a single receptor can bind several chemokines, many chemokines
exhibit a strong tissue and receptor specificity. They play important roles in
inflammation, lymphoid organ development, cell trafficking, cellular
compartmentalization within lymphoid tissues, angiogenesis and wound

healing.

TGF receptors


Receptors for transforming growth factors such as the TGFβ receptor
(Figure 9.4f) possess cytoplasmic signaling domains with serine/threonine
kinase activity.



Signal transduction through cytokine receptors

The ligand-fnduced homo-or heterodimerization of cytokine receptor

subunits represents a common theme for signaling by cytokines. The two
major routes that are utilized are the Janus kinase (JAK)-STAT and the Ras-
MAP kinase pathways. We have already discussed the details of the Ras-
MAP kinase pathway in Chapter 8 (see Figure 8.8) so here we will focus on
the JAK-STAT pathway.

Members of the cytokine receptor superfamily (hematopoi-etin receptors)
lack catalytic domains but are constitutively associated with one or more
JAKs (Figure 9.5). There are four members of the mammalian JAK family:

JAK1, JAK2, JAK3 and Tyk2 (tyrosine kinase 2) and all phosphorylate
their downstream substrates at tyrosine residues. Genetic knockout studies
have shown that the various JAKS have highly specific functions and
produce lethal or severe phenotypes relating to defects in lymphoid

development, failure of erythropoiesis and hypersensitivity to pathogens.

Table 9.2. Chemokines and their receptors. The chemokines are grouped
according to the arrangement of their cysteines (see text). The letter L
designates ligand (i.e. the individual chemokine), whereas the letter R

designates receptors. Names in parentheses refer to the murine homologs of
the human chemokine where the names of these differ, or the murine
chemokine alone if no human equivalent has been described. B, B-cell;

Baso, basophil; DC, dendritic cell; Eosino, eosinophil; MEC, mucosal
epithelial chemokine; Mono, monocyte; Neutro, neutrophil; NK, natural
killer; T, T-cell.

Upon cytokine-induced receptor dimerization, JAKs reciprocally
phosphorylate, and thereby activate, each other. Active JAKs then

phosphorylate specific tyrosine residues on the receptor cytoplasmic tails to
create docking sites for members of the STAT (signal fransducers and

activators of )ranscrip-tion) family of SH2 domain-containing transcription
factors. STATs reside in the cytoplasm in an inactive state but, upon
recruitment to cytokine receptors (via their SH2 domains), become
phosphorylated by JAKs and undergo dimerization and dissociation from

the receptor. The dimerized STATs then translocate to the nucleus where
they play an important role in pushing the cell through the mitotic cycle by
activating transcription of various genes (Figure 9.5). Seven mammalian
STATs have been described and each plays a relatively non-redundant role

in distinct cytokine signaling pathways. Individual cytokines usually
employ more than one type of STAT to exert their biological effects; this is
because the hematopoietin receptors are composed of two different receptor
chains that are capable of recruiting distinct STAT proteins. Further

complexity is achieved due to the ability of STATs to form het-erodimers
with each other, with the result that a single cytokine may exert its
transcriptional effects via a battery of STAT combinations. JAKs may also
act through src family kinases to generate other transcription factors via the

Ras-MAP kinase route (see Figure 8.8). Some cytokines also activate
phosphati-dylinositol 3-kinase (PI3K) and phospholipase C (PLCγ).

Figure 9.5. Cytokine receptor-mediated pathways for gene
transcription.

Cytokine-induced receptor oligomerization activates JAK kinases that are
constitutively associated with the receptor cytoplasmic tails. Upon
activation, JAK kinases phosphorylate tyrosine residues within the receptor
tails, thereby creating binding sites for STAT transcription factors that then

become recruited to the receptor complex and are, in turn, phosphorylated
by JAKs. Phosphorylation of STATs triggers their dissociation from the
receptor and promotes the formation of STAT dimers that translocate to the
nucleus to direct transcription of genes that have the appropriate binding

motifs within their promoter regions. Members of the SOCS family of
inhibitors can suppress cytokine signaling at several points, either through
inhibition of JAK kinase activity directly or by promoting
polyubiquitination and proteasome-mediated degradation of JAKs. The

PIAS family of STAT inhibitors can form complexes with STAT proteins

that either result in decreased STAT-binding to DNA or recruitment of
transcriptional corepressors that can block STAT-mediated transcription.

Cytokine receptors can also recruit additional adaptor proteins such as Shc,
Grb2 and Sos, that can activate the MAP kinase (see Figure 8.8) and PI3
kinase signaling cascades, but these have been omitted for clarity.






































Downregulation of JAK-STAT signaling is achieved by proteins that
belong to the SOCS (suppressor of cytokine signaling) and PIAS ( protein
inhibitor of activated ¿TAT) families (Figure 9.5). SOCS proteins are
induced in a STAT-dependent manner and therefore represent a classical

feedback inhibition mechanism where cytokine signals induce expression of
proteins that dampen down their own signaling cascades. The SOCS family
contains eight members (namely CIS and SOCS1-SOCS7), and these

proteins utilize two distinct mechanisms to downregulate cytokine signals.
On the one hand, SOCS proteins can interact with JAKs, as well as other
signaling proteins such as Vav, and target these proteins for degradation by
the ubiquitin-proteasome pathway (cf. p. 125). Alternatively, SOCS family
proteins can interact with SH2-domain binding sites found within the

activation loop of the JAK kinase domains, thereby blocking access of
JAKs to their downstream substrates (Figure 9.5). Some SOCS family

members, such as CIS ( cytokine- inducible src homology domain 2 [SH2]-
containing), can also directly interact with the STAT-binding SH2 domains

found on cytokine receptors and by doing so can block recruitment of STAT
molecules to the receptor complex. Targeted deletion of SOCS genes in the
mouse has revealed the importance of these proteins for normal cytokine
signaling. SOCS-1-deficient mice display marked growth retardation and

lymphocytopenia and die from inflammation -associated multi -organ
failure within 3 weeks of birth. Consistent with the role of SOCS proteins
as negative regulators of cytokine signaling, lymphocytes derived from
SOCS-1-deficient mice undergo spontaneous activation even in pathogen-

free conditions. SOCS-1-deficient mice generated on a RAG2-deficient
background do not display any of the phenotypes observed on a normal
genetic background, confirming that SOCS-1 exerts its effects primarily
within the lymphocyte compartment.

Thie PIAS family consists of four members (PIAS1, PIAS3, PIASX and
PIASY) and can act to repress STAT-induced tran-scriptional activity by
interacting with these proteins to either restrict their ability to interact with

the DNA promoter elements they associate with, or alternatively, by
recruiting tran-scriptional corepressor proteins such as histone deacetylase
to the STAT transcriptional complexes (Figure 9.5).
JAK-STAT pathways can also be regulated by other mechanisms such as

protein tyrosine phosphatase-mediated antagonism of JAK activity, for
example.



Cytokines often have multiple effects

In general, cytokines are pleiotropic, i.e. exhibit multiple effects on a
variety of cell types (Table 9.1), and there is considerable overlap and

redundancy between them with respect to individual functions, partially
accounted for by the sharing of receptor components and the utilization of
common transcription factors. For example, many of the biological
activities of IL) 4 overlap with those of IL) 13. However, it should be
pointed out that virtually all cytokines have at least some unique properties.

The cytokines produced at the initial stages of T-and B-cell activation
critically influence the subsequent developmental fate of the cell on the

receiving end. Their roles in the generation of T-and B -cell effectors, and in

the regulation of chronic inflammatory reactions (Figure 9.1), will be
discussed at length later in this chapter. We should also note here the

important role of cytokines in the control of hematopoiesis (Figure 9.6).
The differentiation of stem cells to become the formed elements of blood
within the environment of the bone marrow is carefully nurtured through
the production of cytokines by the stromal cells. These include GM)CSF,

G-CSF (granulocyte colony-stimulating factor), M-CSF, IL-6 and -7 and
LIF (Table 9.1), and many of them are also derived from T-cells and
macrophages. It is not surprising therefore that, during a period of chronic
inflammation, the cytokines that are produced recruit new precursors into

the hematopoietic differentiation pathway–c useful exercise in the
circumstances. One of the cytokines, IL-3, should be highlighted for its
exceptional ability to support the early cells in this pathway, particularly in
synergy with IL-6 and G-CSF (Figure 9.6).



Network interactions

The complex and integrated relationships between the different cytokines

are mediated through cellular events. The genes for IL-3, -4 and -5 and
GM-CSF are all tightly linked on chromosome 5 in a region containing
genes for M-CSF and its receptor and several other growth factors and
receptors. Interaction may occur through a cascade in which one cytokine

induces the production of another, through transmodulation of the receptor
for another cytokine and through synergism or antagonism of two cytokines
acting on the same cell (Figure 9.7). Because of the number of
combinations that are possible and the almost yearly discovery of new

cytokines, the means by which target cells integrate and interpret the
complex patterns of stimuli induced by these multiple soluble factors is
only slowly unfolding.

Figure 9.6. Multiple cytokines producted by effector T-cells and other

cells of the immune system can influence hematopoiesis.

Figure 9.7. Network interactions of cytokines.
(a) Cascade: in this example TNF induces secretion of IL-1 and of itself
(autocrine) in the macrophage. (Note that all diagrams in this figure are

simplified in that the effects on the nucleus are due to messengers resulting
from the combination of cytokine with its surface receptor.) (b) Receptor
transmodulation showing upregulation of each chain forming the high
affinity IL-2 receptor in an activated T-cell by individual cytokines and
downregulation by TGFβ. (c) Synergy of TNF and IFNγ in upregulation of

surface MHC class II molecules on cultured pancreatic insulin-secreting
cells. (d) Antagonism of IL-4 and IFNγ on transcription of silent (“sterile”)
mRNA relating to isotype switch (cf. Figure 9.24).

Figure 9.8. Activated T-blasts expressing surface receptors for IL-2
proliferate in response to IL-2

Produced by itself or by another T-cell subset. Expansion is controlled
through downregulation of the IL-2 receptor by IL-2 itself. The expanded
population secretes a wide variety of biologically active cytokines of which
IL-4 also enhances T-cell proliferation.







































Activated T-cells proliferate in response to

cytokines


In so far as T-cells are concerned, clonal proliferation following activation

is critically dependent upon IL-2 (Figure 9.8). This cytokine is a single
peptide of molecular weight 15.5 kDa that acts only on cells that express
high affinity IL-2 receptors (Figure 9.4). These receptors are not present on

resting T-cells, but are synthesized within a few hours after activation.
Activated T-cells divide rapidly for 4–5 days, in an IL-2-dependent manner,
and then differentiate into various effector subsets as we shall discuss
below.

Separation of an activated T-cell population into those with high and low
affinity IL-2 receptors showed clearly that an adequate number of high

affinity receptors were mandatory for the mitogenic action of IL-2. The
numbers of these receptors on the cell increase under the action of antigen
and of IL-2 and, as antigen is cleared, so the receptor numbers decline and,
with that, the responsiveness to IL-2. It should be appreciated that, although

IL-2 is an immunologically nonspecific T-cell growth factor, it only
functions appropriately in specific responses because unstimulated T-cells
do not express high affinity IL-2 receptors.

As we shall see, activated T-cells also produce an impressive array of
other cytokines, and the proliferative effect of IL-2 is reinforced by the
action of IL-4 and, to some extent, IL-6, which react with corresponding
receptors on the dividing T-cells.




Different T-cell subsets can make different


cytokine patterns


We have previously encountered the idea that different types of T-cells can
be generated. Aside from the major subsets of CD4 -and CD8 -restricted T-
cells, further sub -functionaliza-tion of T-cells can be detected on the
basis of the patterns of cytokines that these cells express. As we have

noted earlier, the particular pattern of cytokines secreted by an activated T-
cell is influenced by the nature of the cytokines it is exposed to upon initial
encounter with antigen presented by a mature DC within the secondary

lymphoid organs. In a similar vein, the pattern of cytokines expressed by
DCs are shaped by the nature of the pathogen-associated molecular patterns
(PAMPs) that triggered maturation of the latter, as well as the prevailing
cytokine environment during the initial encounter with the infectious agent.
Switching our attentions back to the T-cell, polarization (i.e. further

differentiation to a particular Th subset) of these cells can be further
reinforced by cytokine signals that are encountered upon trafficking of the
primed T-cell to the site of infection. In this way, T-cell responses can

become tailored to the nature of the pathogen that instigated activation of
the immune system in the first place. However, before we get further into

the details of T-cell polarization, we would caution the reader not to
think of this process as a rigidly constraining one, but rather as a

continuum of responses that can display particularly distinct patterns at
specific points within the spectrum.

Figure 9.9 T-cells can undergo polarization to distinct subsets that
secrete different cytokine combinations.

Naive T-cells can undergo activation and polarization to distinct Th subsets.
Cytokines produced by dendritic cells (DCs) or other innate immune cells,
representing signal 3, dictate the differentiation fate of the T-cell, as shown.






































Th cell polarization


Helper T-cell clones can be divided into three main subsets, Th1, Th2, and
Th17, with each displaying distinct cytokine secretion profiles (Figure 9.9),
which in turn, influences the range of effector functions carried out by each

subset. A further subset of CD4 -positive T-cells has also been identified
that exerts control over the other T-cell subsets by inhibiting their effector
function; such cells are called regulatory T-cells or Tregs. Let us consider
some of the properties that these cytokine profiles confer on their T-cell

subsets.

Th1 cells coordinate responses to intracellular

pathogens


Thi1 cells secrete cytokine profiles skewed towards coordinating responses
to intracellular bacterial and viral infections(Figure 9.9). This is
achieved largely through activating macrophages and assisting the
expansion ofcytotoxic T-lymphocytes (Tc). Because they produce high

amounts of IFNγ, Th1 cells are adept at activating macrophages, which is
particularly important where macrophages have become infected with
intracellular bacteria that actively antagonize macrophage function. When a

Th1 -polarized effector cell arrives at a site of infection, it can be
restimulated by local macrophages that are either infected with intracellular
bacteria or that have internalized bacterial fragments. Presentation of
specific antigen via MHC class II molecules on the macrophage leads to
directed secretion of IFNγ by the Th1 cell to the macrophage (Figure 9.10).

However, in the absence of other signals, macrophages are not very
responsive to IFNγ. This problem is also solved by the Th1 cell in the form
of CD40L, which engages CD40 on the macrophage and greatly increases

its sensitivity to IFNγ. Th1 cells can also enhance the microbicidal
functions of the macrophage to extracellular bacteria that are engulfed via
phagocytosis (Figure 9.10). Recall from Chapter 1 that macrophages greatly
increase their microbicidal properties upon activation and IFNγ as well as
TNFα is a very good way of achieving this. IFNγ-stimulated macrophages

also produce IL-12 that leads to reinforcement of the Th1 phenotype.

Figure 9.10. Th1 cells activate the microbicidal killing activity of
macrophages.
IFNγ derived from Th1 cells is important for the activation of macrophages

and can enhance the microbicidal activity of such cells to kill phagocytosed
bacteria.IFNγ can also induce the secretion of IL-12 and TNF by
macrophages as shown.

Th 1 cells also secrete high levels ofIL-2 (Figure 9.9), which is able to
support the expansion of CD8-positive cytotoxic T-cells, professional
killers of virus-infected cells; we shall discuss how they kill later in this
chapter. This can occur where activated T-cells have migrated to a site of
infection and a Th1 cell engages an infected macrophage or DC (via MHC

class II/peptide-TCR interactions) simultaneously with a CTL, which is
engaged with the APC via MHC class I/peptide-TCR interactions. This
creates the circumstances where a CTL can be induced to clonally expand

to swell its numbers due to IL-2 produced by the Th1 cell. We will see later
in this chapter that a Th1 cell can also “license” a DC for stimulation of a
Tc cell after the Th1 cell has already departed.
Other cytokines secreted by Th1 cells, such as IL-3 and GM-CSF, have

more distant effects on bone marrow precursors and induce the production
of neutrophils and macrophages to swell the ranks of these cells, as
required, during an ongoing infection.


Th2 cells coordinate responses to extracellular

pathogens

Due to their ability to generate IL-4, IL-5 and IL-13 (Figure 9.9), all of
which support B-cell proliferation, class switching and differentiation to

effectors (Figure 9.11), Th2 cells are very good helpers for B-cells and
would seem to be adapted for defense against parasites and other
extracellular pathogens that are vulnerable to IL-4-switched IgE, IL-5-
induced eosinophilia and IL-3/4-stimulated mast cell proliferation. Similar

to Th1 cells, Th2 cells also produce IL-3 and GM-CSF to induce the
production of neutrophils and macrophages from bone marrow precursors.
IL-5 also acts at a distance and is particularly important for production of
eosinophils (Figure 9.1), which, as we discussed in Chapter 1, are

particularly well adapted towards combating large extracellular parasites
such as parasitic worms. Due to their physical size, these infectious agents
cannot be readily phago-cytosed by macrophages or neutrophils. To deal
with this problem, eosinophils are equipped with specialized granules

containing a range of cytotoxic molecules that are released onto the surface
of the parasite upon engagement of the complement C3b receptors on the
eosinophil with C3b-opsonized parasites.

Figure 9.11. B-cell response to thymus-dependent (TD) antigen: clonal

expansion and maturation of activated B-cells under the influence of T-
cell-derived soluble factors.
Co-stimulation through the CD40L-CD40 interaction is essential for
primary and secondary immune responses to TD antigens and for the

formation of germinal centers and memory. c-myc expression, which is
maximal 2 hours after antigen or anti-| stimulation, parallels sensitivity to
growth factors; transfection with c-myc substitutes for anti-|μ.

Th17 cells promote acute inflammatory responses

and recruit neutrophils


A relatively recent addition to the T-helper cell fold, Th17 cells are
currently receiving considerable attention for their propensity to be
involved in autoimmune reactions when the actions ofthese cells get out
ofcontrol. Th17 cells are IL-17A-producing cells that also secrete IL-17F,

IL-21 and IL-22 (Figure 9.9). These cells appear to be specialized towards
mounting massive inflammatory responses towards extracellular bacterial
and fungal infections, particularly at mucosal interfaces. This appears to
be achieved through production of IL-17A, IL-17F and IL-22, which have

broad effects on many nonimmune cell types, such as endothelial and
epithelial cells, and elicit the production of proinflammatory cytokines and
chemokines by such cells to promote neutrophil recruitment to the site of
inflammation. These cytokines also induce the secretion of antimicrobial

peptides, by keratinocytes for example, which strengthens their barrier
function towards infection.

Cross-regulation of Th1, Th2 and Th17 subsets


Not only do the particular cytokine secreted by Th1, Th2 and Th17 cells
enable them to elicit distinct biological functions, these cytokines also help
to reinforce the same pattern of cytokine production, as well as inhibiting
polarization to the alternative Th subset, a feature that is sometimes

exploited to the benefit of certain pathogens. The ability of IFNγ, the
characteristic Th1 cytokine, to inhibit the proliferation of Th2 clones, and of
Th2-derived IL-4 and -10 to block both proliferation and cytokine release
by Th1 cells, would seem to put the issue beyond reasonable doubt (Figure

9.12). Similarly, development of the Th1 or Th2 phenotype appears to be
antagonistic to the development of Th17 cells.
Studies on the infection of mice with the pathogenic protozoan

Leishmania major demonstrated that intravenous or intraperitoneal injection
of killed promastigotes leads to protection against challenge with live
parasites associated with high expression of IFNγ mRNA and low levels of
IL-4 mRNA; the reciprocal finding of low IFNγ and high IL-4 expression

was made after subcutaneous immunization that failed to provide
protection. Furthermore, nonvaccinated mice infected with live organisms
could be saved by injection of IFNγ and anti-IL-4. These results are
consistent with the preferential expansion of a population of protective

IFNγ-secreting Th1 cells by intraperitoneal or intravenous immunization,
and of nonprotective Th2 cells producing IL-4 in the subcutaneously
injected animals.


Stability versus plasticity of Th subsets


The original Mosmann–Coffman classification into Th1 and Th2 subsets
was predicated on data obtained with clones that had been maintained in
culture for long periods and might have been artifacts of conditions in vitro.
Thie use of cytokine-specific monoclonal antibodies for intracellular

fluorescent staining, and of ELISPOT assays (cf. p. 178) for the detection of
the secreted molecules, has demonstrated that the Th1 and Th2 phenotypes
are also apparent in freshly sampled cells and thus also applies in vivo.

Nonetheless, it is perhaps best not to be too rigidly constrained in one’s
thinking by the Th1/Th2/ Th17 paradigm, but rather to look upon activated

T-cells as potentially producing a whole spectrum of cytokine profiles (Th0,
Figure 9.12), with possible skewing of the responses towards particular

patterns depending on the nature of the antigen stimulus. Thus, other
subsets may also exist, in particular the transforming growth factor-β
(TGFβ) and IL-10-producing Th3/Tr1 (T-regulatory 1) cells, which are of
interest because these cytokines can mediate immunosuppressive effects

and may be involved in the induction of mucosally induced tolerance (cf. p.
508). Another subset that is also emerging in the scientific literature is a
class of Th cells that have been dubbed follicular helper T cells (Tfh) that
appear to be important for guiding B-cell development, class switching and

survival within germinal centers. Tfh cells have been found to produce high
levels of IL-21 and to migrate to the follicular regions upon activation to
form stable contacts with antigen-primed B cells.

Figure 9.12. The generation of Th1 and Th2 CD4 subsets.

Following initial stimulation of T-cells, a range of cells producing a
spectrum of cytokine patterns emerges. Depending on the nature of the
pathogen and the response of cells of the innate immune system during the
initial stages of infection, the resulting T-helper cell population can be

biased towards two extremes. Th1-promoting pathogen products (such as
LPS) engage Toll-like receptors (TLRs) on dendritic cells (DCs) or
macrophages and induce the secretion of Th1-polarizing cytokines such as
IL-12 and IL-27. The latter cytokines promote the development of Th1 cells

that produce the cytokines characteristic of cell-mediated immunity. IL-4,
possibly produced by interaction of microorganisms with the lectin-like
+
NK1.1 receptor on NKT-cells or through interaction of Th2-promoting
pathogen products with TLRs on DCs, skews the development to the
production of Th2 cells whose cytokines assist the progression of B-cells to

antibody secretion and the provision of humoral immunity. Cytokines
produced by polarized Th1 and Th2 subpopulations are mutually inhibitory.
LT, lymphotoxin (TNFp); Th0, early helper cell producing a spectrum of
cytokines; other abbreviations as in Table 9.1.

It is very likely that further T-cell subsets will be identified in the coming
years and current evidence suggests that rather than each subset
representing highly committed and distinct T-cell “lineages,” it seems that
there is considerable plasticity in the spectrum of cytokines that

differentiated T-cells can secrete. Furthermore, it is also apparent that
reprogramming of effector T-cells can occur, converting differentiated T-
cell subsets from one type towards another.




Cells of the innate immune system shape

the Th1/Th2/Th17 response


We have already introduced the concept that the cytokine milieu that

becomes established by cells of the innate immune system during the early
stages of infection has a major influence on the adaptive immune response
(Figures 9.2 and 9.12). In the initial stages of an infection, the innate

immune responses hold the line as T-lymphocytes require priming by DCs
to initiate clonal expansion and maturation to effectors. Upon migration of
antigenttpecific T-cells to lymph nodes where they come in contact with
mature DCs fresh from their encounters with microbial pathogens, the
pathogen products encountered by the DC will have polarized the latter in

favor of secreting particular cytokines, as we have discussed above (Figure
9.9). Polarization ofT-cells towards a Th1, Th2 or other fate is achieved via
signal 3 and the nature of this signal is strongly influenced by the conditions

under which the APC is primed (Figure 9.12).

Th1 polarization


IL-12 and its relatively recently discovered relatives, IL-23 and IL-27, are
instrumental in polarizing towards a Th1 cell phenotype (Figure 9.12).
Invasion of phagocytic cells by intracellular pathogens induces copious
secretion of IL-12, which in turn stimulates IFNγ production by NK cells.

Engagement of many of the known Toll-like receptors (TLRs) on DCs by
microbial products (such as LPS, dsRNA and bacterial DNA) triggers DC
maturation and induces IL-12 production, thereby favoring Th1 responses.
Bacterial priming also induces CD40 receptor expression on DCs and

induces responsiveness to CD40L, expressed by activated T-cells, for
optimal IL-12 synthesis. IL-12 is also particularly effective at inducing
IFNγ by activated Ttiells and secretion of the latter by the T-cell further
enhances IL-12 production and secretion by DCs; this acts as a classical

positive feedback loop for enhancement of IL-12 production and further
skews the response towards Th1.


Th2 polarization


IL-4 is pivotal for the production of a Th2 cell phenotype. While IL-12 and
IFNγ promote a Th1 response, these cytokines also inhibit Th2 responses
(Figure 9.12). However, IL-4 effects appear to be dominant over IL-12 and

therefore the amounts of IL-4 relative to the amounts of IL-12 and IFNγ
will be of paramountimportance in determining the differentiation of Th0
(i.e. unpolarized) cells into Th1 or Th2. IL-4 downregulates the expression

of the ILi 12R β sub unit necessary for responsiveness to IL-12, further
2
polarizing the Th2 dominance. It is still unclear whether signals from the
innate immune system drive T-cells in the direction of a Th2 response or

whether this is a default differentiation pathway for Th cells unless
suppressed by Th1-polarizing signals such as IL-12 or IFNγ A special cell
+
population, the NKT-cells bearing the NK1.1 marker, rapidly releases an
IL-4-dominated pattern of cytokines on stimulation. These cells have many
+ -
- -
unusual features. They may be CD4 8 or CD4 8 and express low levels of
T-cell αβ receptors with an invariant α chain and very restricted β many of

these receptors recognizing the nonclas-sical MHC-like CD1 molecule.
Their morphology and granule content are intermediate between T-cells and

NK cells. Although they express TCR αβ there is an inclination to classify
them on the fringe of the “innate” immune system with regard to their

primitive characteristics and possession of the lectin-like NK1.1 receptor
that may be involved in the recognition of microbial carbohydrates.


Th17 polarization


Although the precise cocktail of cytokines that triggers the production of
Th17 cells is still a matter of active debate, it is clear that the pro-
inflammatory cytokine IL-6 plays a particularly influential role initially.

This is then reinforced by IL-23, which appears to be important for
expansion and stabilization of these cells (Figure 9.9). Naive T cells do not
express IL-23 receptors, but upregulate these upon productive activation,
which is also enhanced by IL-6. Thus, the role of IL-23 in differentiation to

Th17 cells is one of reinforcement rather than initiation. There is also
evidence that TGFβ in combination with IL-6 influences the generation of
Th17 cells, whereas TGFβ alone polarizes T-cells towards a Treg fate, as
we shall discuss below. However, TGFβ does not appear to play an

instructive role for the production of Th17 cells, rather, it appears to act by
suppressing the development of either the Th1 or Th2 phenotypes, which
are antagonistic to the Th17 fate.



Further thoughts on Th polarization

Whilst there is a certain amount of evidence indicating the existence of

subpopulations of dendritic cells specialized for the stimulation of either
Th1 or Th2 populations, it seems that DCs are relatively plastic and can
adopt a Th1 -, Th2 -or Th17-polarizing phenotype depending on the
priming signals they encounter from microbial and tissue-derived sources.
However, it should be obvious from the above discussion that the cytokines

produced in the immediate vicinity of the T-cell will be important.



Policing the adaptive i mmune system


In addition to the effector T-cell subsets that we have already discussed,

there is also much evidence that T-cells can also differentiate into cells that

play a suppressive or regulatory role in immune responses (Figure 9.9).
That is to say, these cells appear to police the actions of the other classes of

T-cells, stepping in to quell immune responses when this appears necessary.
Such cells are called regulatory T-cells, or Tregs, and there appear to be two
different categories of such cells, natural and inducible T-regs. These cells
play a role in suppressing responses to self antigens, as well as

inappropriate or undesirable responses to nonself antigens (such as
commensal bacteria or food in the gut); indeed, it is now believed that Tregs
control almost every adaptive immune response. We shall look at natural
Tregs first as these appear to be the most abundant type.



Natural Tregs

+
+
Natural or thymic-derived Tregs are a population of Foxp3 CD25 CD4 +
T-cells that can suppress immune responses of autoreactive T-cells by

mechanisms that are still not entirely understood, but appear to involve
several distinct and possibly overlapping strategies (see below). The current
view is that these self) antigen” )eactive T cells develop in the thymus and
are released as functionally mature cells that can act to dominantly suppress

the activation of other self-reactive T-cells that escape negative selection in
the thymus, possibly through competition for self-antigens presented by
APCs or through CTLA4-mediated signals from the Treg to the APC.
Natural Tregs constitute 5–10% of CD4-positive T-cells and their

development is critically dependent on the induction of Foxp3, a
transcription factor that can repress the transcription of Th1-, Th2-and Th
17-type cytokines. Loss-of-function mutations in the FOXP3 gene result in

a variety of inflammatory and autoimmune defects characterized by
massive overproduction of Th1-and Th2-type cytokines, which is ultimately
fatal. Tregs appear to be essential for the ongoing suppression of
autoreactive T-cells, as their depletion results in the spontaneous
development of autoimmune disease in otherwise normal mice. In humans,

the equivalent condition resulting from mutations in the gene encoding
Foxp3 is known as immune dysregulation, polyendocrinopathy,
enteropathy, X-linked (IPEX). Autoimmune disease can also be provoked

by adoptive transfer of Treg-depleted splenocytes from normal adult mice
to syngenic recipients lacking T-cells. In vitro stimulation of Foxp3+Treg)